The present invention relates generally to a system, method, and apparatus for determining the concentration of a substance of interest in a fluid sample. More particularly, the invention relates to a copolymer-based immunoassay system and method and apparatus coacting to quantitatively measure the concentration of biologically active substances capable of promoting or inhibiting agglutination reactions.
The medical community has a continuing need for improved procedures for measuring the concentration of biologically active substances, e.g., antigens and antibodies, in body fluids. Such procedures are needed, for example, to measure the concentration of drugs, hormones, and other substances in blood, serum, plasma, urine, and other body fluids as an aid in medical diagnosis and in the administration of drugs.
Various immunoassay procedures have been developed to determine the presence of biologically active substances in fluid samples. Perhaps the dominant immunoassay approach in use today is radioimmunoassay (RIA). In this method, a sample containing an unknown concentration of antigen is mixed with a fixed amount of antibody and a fixed amount of antigen which has been radioactively labelled. The resultant antibodyantigen complex is recovered and its radioactivity measured to determine the relative concentrations of the radioactive antigen and the antigen contributed by the sample to determine the concentration of the antigen in the sample.
Radioimmunoassay is popular because of its relatively high sensitivity. It suffers, however, from many well-known limitations including a relatively short shelf life, the necessity of handling radioactive materials, and the disposal problems associated with such materials. Also, radioimmunoassay systems are relatively expensive and, thus, are generally used only in large clinics and hospitals where there is sufficient testing volume to make the equipment cost effective. Radioimmunoassay methods are also generally not used for measuring antibody concentrations and are usually used in applications in which antigen concentrations are to be measured.
Enzyme immunoassay has replaced radioimmunoassay in some segments of the market; and although enzyme immunoassay is gaining in popularity, the sensitivity of this technique is still limited. In addition, the sophistication of the instrumentation and the resultant high skill level required of the technician would also appear to limit this procedure to hospitals and other large facilities.
Agglutination immunoassays have been in use for over thirty years. The agglutination reaction involves the in vitro aggregation of microscopic carrier particles as a result of the specific reaction between antibodies and antigens, one of which is immobilized on the surface of carrier particles. Early systems used red blood cells as carrier particles, while more recent systems also utilize latex-based particles.
Agglutination immunoassays are performed in two basic formats, direct and indirect. In the direct format, carrier particles are sensitized by attaching a specific antibody to the surfaces of the carrier particles. A fluid test sample is then mixed with a suspension of the sensitized carrier particles, and if the corresponding antigen is present in the test sample, the particles agglutinate through the resulting antibody-antigen binding reaction. By detecting the agglutination reaction, the presence of the antigen in the test sample can be confirmed.
Alternatively, an antigen can be attached to the surfaces of the carrier particles to detect the presence of the corresponding antibody in the test sample.
In the indirect format, to the extent to which a quantity of antibody (or antigen) of interest is present in a test sample, the antibody (or antigen) of interest inhibits the aggregation of the carrier particles that would otherwise have occurred; and the degree of inhibition indicates the concentration of the antigen (or antibody) of interest in the test sample.
The agglutination test is typically performed on a slide and read visually, and is a highly subjective test requiring substantial expertise. When no agglutination occurs, the mixture appears homogeneous and milky (when latex particles are used as the carrier particles); whereas, when aggregation occurs, the latex particles initially appear granular and, with time, become clumped. Because of the generally subjective nature of interpreting the results of the reaction, it is difficult to extract quantitative information, making the process unsatisfactory in many applications, for example, to monitor hormone levels or to detect cancer markers.
Because of the highly qualitative and subjective nature of prior agglutination immunoassay systems, various efforts have been directed toward developing systems capable of providing a more quantitative measurement. In addition, it has long been a desideratum to provide very sensitive immunoassay systems that are not dependent on large expensive apparatus and complicated and sensitive sample preparation procedures.
In U.S. Pat. No. 4,721,681 to Lentrichia et al., for example, an agglutination immunoassay system is described in which a sample containing, for example, an antigen of interest is reacted with a first reagent containing antibody-coated, light particles and a second reagent containing antigen-coated, heavy particles in a centrifugal field. Differential migration of first particles, second particles, and first particles linked to second particles by an agglutination reaction leaves a concentration of particles in suspension at a locus over time, which concentration is a function of the concentration of the antigen of interest in the test sample.
U.S. Pat. No. 4,264,766 to Ernst A. Fischer describes a latex polymer and process for its manufacture to provide improved latex particles for agglutination immunoassay tests. In the Fischer patent, latex carrier particles with a size range of about 0.01 microns to about 0.9 microns and a specific gravity of about 1.0 are provided with a covalently bound, water-soluble polyhydroxy compound capable of covalently binding an immuno active agent. The latex particles are manufactured in an aqueous latex suspension by reacting the suspended latex particle, having reactive functional groups selected from a group consisting of carboxyl, amino, amido or nitrile groups, with a water-soluble polyhydroxy compound.
U.S. Pat. Nos. 4,118,192; 4,203,724; and 4,208,185 to Sawai et al. describe an agglutination immunoassay system in which antibody- or antigen-sensitized latex particles, with an average diameter not greater than 1.6 microns, are irradiated with light (substantially monochromatic or polychromatic) having a wavelength in the range of 0.6 to 2.4 microns and longer than the average diameter of the carrier particles by a factor of at least 1.5, and the absorbance of the reaction mixture is measured as the reaction takes place. The described system uses insoluble carrier particles such as lattices of organic polymers such as polystyrene and styrene butadiene copolymer, dispersed coccal bacteria as well as microparticles of inorganic oxides and finely pulverized minerals, metals, and the like. Light sources may include tungsten lamps (monochromatically filtered or unfiltered), xenon lamps, halogen lamps, the Nernst glower, nichrome wire, or light-emitting diodes, with Ga-As light-emitting diodes being particularly favored. Light is split and transmitted through the reaction product and through a control sample for compensation purposes and is measured by two photocells such as those having lead sulfide, photoconductive elements. The photocell's outputs were amplified and recorded with time to provide data. Such systems have been incorporated by Mitsubishi Chemical Industries Limited into large hospital immunoassay facilities, but have failed to provide sensitivities in the range of 10.sup.-6 grams/milliliter to 10.sup.-12 grams/milliliter.
U.S. Pat. No. 4,080,264 to Cohen et al. discloses an agglutination immunoassay procedure which utilizes light-scattering spectroscopy to determine the concentration of an antibody or antigen of interest in a test sample. In Cohen et al., the mean diffusion constant of the agglutination reaction product is examined by quasi-elastic light-scattering spectroscopy and compared with predetermined mean diffusion constants to provide a quantitative measure of the concentration of the antibody or antigen of interest in the test sample. In Cohen et al., the light source is a laser. Such components are relatively expensive to purchase and maintain, provide errant outputs, and render the system useful in only relatively high-volume facilities. Such systems have been shown to have a substantial lack of sensitivity and are unable to accurately detect an agglutination reaction unless a high degree of agglutination has taken place, i.e., there is a substantially high concentration of the substance of interest.
U.S. Pat. No. 4,174,952 of Cannell et al. describes an agglutination immunoassay system in which the ratio of the intensity of light scattered at two different angles is measured and compared with standard measurements of known concentrations. This system has also been shown to have an unsatisfactory sensitivity and is too expensive for use in private physicians' offices or small labs.